The eukaryotic proteasome is a cylindrical multisubunit complex that contains a buried central chamber in which intracellular proteins are degraded. In 1992, we discovered two particles that bind the ends of the proteasome and markedly activate peptide hydrolysis presumably by opening channels to the central chamber. One of these activators, the 11S REG, is a heptameric ring containing alpha and beta subunits. Over the past six years, we have cloned and expressed cDNAs encoding REGalpha, REGbeta and a homologous activator, REGgamma. We have characterized the recombinant proteins and a variety of REGalpha, mutants. We now propose additional mutagenesis experiments to assign functions for regions within each REG homolog and to generate dominant-negative mutants. Our collaborator, Chris Hill, has solved the crystal structure of the REGalpha heptamer. It is a conical ring with a central, solvent-filled channel. The "lower" surface of the REGalpha, ring binds the proteasome. Unresolved on the "upper" surface of each subunit are 39 residues that encompass sequences unique to each REG homolog. In REGalpha this unique stretch of amino acids consists of 28 "alternating" lysine (K) and glutamate (E) residues. We call such regions "KEKE motifs", and they are enriched in proteasome subunits and in chaperones. Using yeast two hybrid screens, GST-REG chimeras and REG homologs derivatized with radioiodinated photoreactive crosslinkers, we will identify proteins that bind the "upper" surfaces of the REGalpha/beta and the REGgamma heptamers. There is circumstantial evidence that REGalpha/beta heptamers play a role in Class I antigen presentation. We will test this idea directly by over expressing wild-type or dominant negative REGalpha and REGbeta mutants in cultured mouse cells and measuring the surface expression of an ovalbumin Class I epitope. Because KEKE motifs are also enriched in precursors to peptides presented on Class I molecules, we hypothesize that they may actually promote presentation of peptides. We will test this hypothesis by producing plasmids that encode KEKE or non-KEKE regions N-terminal to the ovalbumin epitope. The precursors will be expressed in mouse LKb cells, and the surface expression of Class I-ovalbumin peptide complexes will be measured using a quantitative monoclonal antibody assay.